Characterization of the complete mitochondrial genome of the Libelloides sibiricus (Neuroptera, Ascalaphidae)

Abstract Libelloides sibiricus (Eversmann, 1850) is widely distributed in China, Korea and eastern Russia. To date, few studies have been conducted on this species, with the exception of morphological taxonomy studies. In this study, we sequenced the complete mitochondrial genome (mitogenome) of Libelloides sibiricus, which is 15,811 bp in length, with an overall A + T content of 74.8%, encoding 2 ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding genes, and a control region. The gene arrangement and components of L. sibiricus are identical to those of most other Neuropteran species. TAA is utilized as the termination codon for most PCGs and TAG for nd1, however, nd6 and atp6 used the incomplete termination codon TA- and cox1, cox2, nd5, cytb had termination codons consisting of only T–. In addition, we selected all known 59 species of Neuroptera from NCBI, and used Sialis hamata, Sialis melania, Sialis longidens and Sialis jiyuni (Megaloptera: Sialidae) as the outgroup. Phylogenetic analysis suggested that the mitogenome of L. sibiricus was the most closely related to L. macaronius and all the owlflies formed the monophyletic group within the superfamily Myrmeleontoidea.


Introduction
Owlfly, which is the common name for the insect group known as Ascalaphidae, has large adult individuals with long, elongated antennae that are enlarged at the tips.The larvae, on the other hand, are very flat and have branched spines and decorative hairs on their body surface.In 1842, Lef� ebvre established the family Ascalaphidae, which includes two subfamilies: Holophthalminae and Ascalaphinae.In recent years, there have been significant changes to the understanding of the phylogenetic status of the family Ascalaphidae (Jones 2019;Machado et al. 2019).It proposes that the family Ascalaphidae may actually be a subfamily within the larger family Myrmeleontidae.These revised taxonomic considerations shed new light on the evolutionary history of Ascalaphidae and provide an opportunity for a comprehensive reevaluation of its classification.Future studies can now focus on exploring the genetic relationships and characteristics of these insects to confirm and refine these taxonomic revisions.However, to date, only five owlflies species mitochondrial genomes have been fully sequenced.There are Libelloides macaronius (NC_015609), Ascaloptynx appendiculatus (FJ171324), Ascalohybris subjacens (KC758703), Ogcogaster segmentator (ON243766), Suhpalacsa longialata (MH361300).The genus Libelloides , a member of the family Ascalaphidae, subfamily Ascalaphinae includes 16 species and subspecies occurring in Palacarctic region (H� ava 2000;H� ava and� Abrah� am 2014, Asp€ ock et al. 2001).In this study, we aimed to sequence and characterize the entire mitogenome of Libelloides sibiricus to provide a complete mitogenome reference that is valuable for determining robust phylogenetic relationships and population genomics in owlflies.

Materials and methods
The material of Libelloides sibiricus (Figure 1a,b,c) was collected from Xianghetun, Gaojiadian Town, Xifeng County, Liaoning Province (Geographic location: 42 � 67'13"N, 124 � 45'92" E), China, on 26 May.2023.The sample was alive during the collection and the specimen was deposited in the Museum of Nanjing Police University under the voucher number NFPC0146 (Qingbin Zhan, zhanqb@nfpc.edu.cn).Total genomic DNA was extracted from thoracic muscle of adult moths using the cetyltrimethylammonium bromide (CTAB) method (Shahjahan et al. 1995).The DNA quality was examined with agarose-gel electrophoresis, and the concentration was measured using Nanodrop.Paired-end libraries with insert sizes of 180 bp were prepared from 1 mg DNA of genomic using Illumina 's Genomic DNA Sample Preparation kit (Illumina, San Diego, CA, USA), following the manufacturer's instructions.Subsequently, the DNA libraries were sequenced using 150 bp paired reads on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA).Fastp was used to check the quality of data (Chen et al. 2018).A total of 50,504,036 clean reads were generated.Quality control standards are as follows: first, the reads with unrecognized nucleotides >10% were removed.Second, removing reads with >50% bases having Phred quality <5, and the readings aligned with the adapter greater than 10 nt were removed, allowing mismatches � 10%.Finally, the assumed PCR repeats generated by PCR amplification during library construction were removed (the same readings 1 and 2 in the two paired end readings).The mitochondrial genome of L. sibiricus was assembled using Novoplasty v2.7 (Nicolas et al. 2016).The MITOS2 web server (Bernt et al. 2013) was utilized to annotate the complete mitogenome of L. sibiricus.The tRNA genes were annotated using tRNAscan-SE software (Lowe and Eddy 1997), To ensure accuracy, the annotation errors in the mitochondrial genome were manually corrected using Apollo software [Lewis et al. 2002].The drawing of mitogenome map was completed by Proksee (Grant et al. 2023).
To investigate the phylogenetic position of L. sibiricus, we selected all known 59 species sequences of Neuroptera from NCBI, and used Sialis hamata, Sialis melania, Sialis longidens and Sialis jiyuni (Megaloptera: Sialidae) as the outgroup.The protein-coding sequences were aligned using MAFFT version 7.313 (Katoh and Standley 2013).After removing gaps and ambiguous sites using trimAI (Capella-Guti� errez et al. 2009), we concatenated the sequences using PhyloSuite version 1.2.2 (Zhang et al. 2020).A phylogenetic tree was constructed based on the mitochondrial genome, including 13PCGs and 2 rrna dataset, and the maximum-likelihood (ML) method with the best-fit model estimated using IQ-TREE v1.6.10 (Nguyen et al. 2015).Finally, 1000 bootstrap replicates were used, and the phylogenetic trees were visualized and annotated using the Interactive Tree of Life (ITOL) (https://itol.embl.de/).

Results
The complete circular mitogenome of L. sibiricus presented 15881 bp in size (GenBank accession no.OR571471).It contains 37 genes (13 PCGs, 22 tRNA genes, and two rRNA genes) and a control region (Figure 2).The mitogenome sequence has a high AT content of 74.8%.In addition, the majority coding strand (J-strand) contains 23 genes (nine PCGs and 14 tRNA genes) and the minority coding strand (Nstrand) contains 14 genes (four PCGs, eight tRNA genes, and two rRNA genes).Analysis of the PCGs showed that the total length of the 13 PCGs was 11,177 bp, of which the nad5 gene was the longest of 1732 bp and atp8 was the shortest of 159 bp.All 13 PCGs initiated with the standard start codons ATN, excpt cox1 with ACG (Table 1).Whereas seven PCGs terminated with complete stop codons (i.e.TAA and TAG), nd6 and atp6 use the incomplete termination codon TA-and cox1, cox2, nd5, cytb used a single T-residue as the stop codon.The analysis of rRNA genes showed that the length of 16S rRNA was 1315 bp with the AT content of 78.1%, and the length of 12S rRNA was 776 bp with the AT content of 74.7%.
To understand the evolutionary status of L. sibiricus, the mitochondrial genome of 59 species of Neuroptera and four outgroup species of Sialidae were used for phylogeny construction.The maximum likelihood-based phylogenetic tree revelaed (Figure 3) that the mitogenome of L. sibiricus is the most closely related to L. macaronius with 100% bootstrap support, as shown in Figure 3. Furthermore the phylogenetic tree of the mitochondrial genome of 59 species of Neuroptera revealed that all the owlflies(Ascaloptynx appendiculatus, Suhpalacsa longialata, Ogcogaster segmentator, Ascalohybris subjacens, L. macaronius and L. sibiricus) formed the monophyletic group within the superfamily Myrmeleontoidea.

Discussion and conclusion
Hypotheses of the exact relationship of the Ascalaphidae and Myrmeleontidae to the other families of the Myrmeleontiformia have changed with time.The paraphyly of Myrmeleontidae, with the previously nested Ascalaphidae, has been supported by multiple phylogenomic studies (Wang et al. 2016, Winterton et al. 2017, Machado et al. 2019).Specifically, Machado et al. (2019) proposed a new classification of Myrmeleontidae, treating Ascalaphidae as a subfamily.In this study, we present the complete mitochondrial genome of L. sibiricus.Overall, our findings are consistent with those of Jones (2019) and Machado et al. (2019).However, current molecular data for Ascalaphidae are very limited, especially mitochondrial genome-wide data, which may help resolve the taxonomic inconsistency between the molecular and morphological identification of species in this family more accurately.

Figure 2 .
Figure 2. Circular map of the mitochondrial genome of L. sibiricus.Genes outside the circle are encoded on the heavy strand and genes inside the circle are encoded on the light stand.The inner black bars indicate the GC content, and the Middle line represents 50%.visualization was performed using proksee (Grant et al. 2023).

Figure 3 .
Figure 3. Based on the PCG12 matrix, the phylogenetic tree was reconstructed under the maximum-likelihood (ML) methods, with branch support values denoted SH-Alrt/UFBoot2.The mitochondrial genomes of 59 species of neuroptera and four outgroup species of Sialidae (S. hamata, S. melania, S. longidens and S. jiyuni)